Levels of metallothionein messenger RNA in foetal, neonatal and maternal rat liver

We have used translation in vitro of hepatic polyadenylated RNA to characterize the levels of metallothionein mRNA in foetal, neonatal, pregnant and nulliparous rats. The translation products of foetal hepatic metallothionein mRNA increased relative to other mRNA translation products from day 18 of gestation to birth and attained a maximum, maintained throughout suckling, which is tenfold above 17-day foetal hepatic levels and fourfold above adult levels. Maternal liver metallothionein mRNA decreased fivefold between 17 days and 20 days of gestation, rose sharply immediately before birth, and was low throughout lactation.     

Variation in the amounts of hepatic copper, zinc and metallothionein mRNA during development in the rat.

Amounts of hepatic metallothionein mRNA were assessed in RNA from foetal and neonatal rat livers by using dot-blot hybridization. Metallothionein mRNA began to increase about day 15 of gestation and reached a foetal maximum of 5-fold higher than adult values between 18 and 21 days of gestation. The amounts fell significantly for the first 3 days after parturition, and rose again to 6-fold above adult values 6 days after birth. By 15 days after birth the metallothionein mRNA had declined to adult amounts. In comparison, amounts of ornithine transcarbamoylase mRNA did not vary greatly during development. Hepatic zinc concentrations increased from day 14 of gestation to a maximum just before birth, and remained above adult values until 30 days after birth. From 14 days of gestation to 8 days after birth, hepatic copper concentrations were about 4-fold higher than in the adult, but a substantial increase (to about 9-fold higher than in the adult) occurs between 10 and 15 days after birth. CdCl2 administered to pregnant rats on day 18 of gestation was shown to block placental transfer of zinc, and we found decreased foetal hepatic zinc concentration after the CdCl2 treatment, but this failed to cause a significant decrease in metallothionein mRNA, suggesting that zinc may not be the primary inducer of hepatic metallothionein mRNA during foetal life.     

Phosphoenolpyruvate carboxykinase and pyruvate carboxylase in developing rat liver

1. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase were measured in foetal, newborn and adult rat liver extracts by a radiochemical assay involving the fixation of [¹⁴C]bicarbonate. 2. Pyruvate-carboxylase activity in both foetal and adult liver occurs mainly in mitochondrial and nuclear fractions, with about 10% of the activity in the cytoplasm. 3. Similar studies of the intracellular distribution of phosphoenolpyruvate carboxykinase show that more than 90% of the activity is in the cytoplasm. However, in the 17-day foetal liver about 90% of the activity is in mitochondria and nuclei. 4. Pyruvate-carboxylase activity in both particulate and soluble fractions is very low in the 17-day foetal liver and increases to near adult levels before birth. 5. Phosphoenolpyruvate-carboxykinase activity in the soluble cell fraction increases 25-fold in the first 2 days after birth. This same enzyme in the mitochondria has considerable activity in the foetal and adult liver and is lower in the newborn. 6. Kinetic and other studies on the properties of phosphoenolpyruvate carboxykinase have shown no differences between the soluble and mitochondrial enzymes. 7. It is suggested that the appearance of the soluble phosphoenolpyruvate carboxykinase at birth initiates the rapid increase in overall gluconeogenesis at this stage.     

Development of gluconeogenesis in neonatal rat liver. Effect of triamcinolone

1. The normal development of the key enzymes of gluconeogenesis in rat liver, glucose 6-phosphatase, hexose diphosphatase, phosphopyruvate carboxylase and pyruvate carboxylase, was measured during the neonatal period. 2. Glucose 6-phosphatase, hexose diphosphatase and pyruvate carboxylase are all present in the late foetal liver, but all the enzymes show an increase in activity after birth. 3. Phosphopyruvate carboxylase is not present in liver extracts from foetal rats, but activity appears immediately after birth and increases rapidly over the first day and then more slowly to reach its maximum at the fourth postnatal day. 4. The fluorinated synthetic glucocorticoid, triamcinolone, was administered to foetal rats at various gestation times by intraperitoneal injection in utero and the animals were killed at intervals between 4 and 48hr. later. 5. The administration of triamcinolone results in slight depression of glucose 6-phosphatase, and a more significant depression of hexose diphosphatase to about one-half its normal activity in foetal rat liver. 6. Triamcinolone injection is without effect on pyruvate carboxylase activity and does not result in premature appearance of phosphopyruvate carboxylase in foetal rat liver. 7. Pyruvate kinase and aspartate amino-transferase activities in foetal rat liver are both depressed by triamcinolone treatment, whereas phosphofructokinase activity is elevated. 8. Tyrosine amino-transferase activity in foetal rat liver is markedly elevated in animals exposed to triamcinolone for 10hr. or more, but the effect is only observed in animals close to term. 9. The results are discussed in relation to mechanisms involved in the initial synthesis of tissue-specific enzymes in developing tissues, and it is concluded that glucocorticoids do not initiate the synthesis of the gluconeogenic enzymes.     

Localization of immunoreactive transthyretin (prealbumin) and of transthyretin mRNA in fetal and adult rat brain

We used a combination of immunohistochemical and molecular-biological techniques to investigate the localization of transthyretin (TTR) in the brains of adult and fetal rats. The immunohistochemical studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of TTR using the unlabeled peroxidase-antiperoxidase method. TTR mRNA levels were measured by Northern-blot analysis of poly (A+) RNA, followed by hybridization to 32P-labeled TTR cDNA; TTR mRNA was localized in brain tissue sections by in situ hybridization. Immunoreactive TTR was found to be specifically localized in the choroid plexus epithelial cells of adult rat brain. High levels of TTR mRNA were found in poly (A+) RNA samples obtained from the choroid plexus. In addition, the specific localization of TTR mRNA in the epithelial cells of the choroid plexus was demonstrated by in situ hybridization. Neither immunoreactive TTR nor TTR mRNA were found in other regions of adult rat brains. The levels of TTR mRNA in the choroid plexus were at least 30 times higher than those observed in the adult liver. Immunoreactive TTR was observed in the brains of fetal rats on as early as the 11th day of gestation. This immunoreactive TTR was localized in the tela choroidea, the developmental forerunner of the choroid plexus. Immunoreactive TTR was also observed in the fetal choroid plexus as it began to form (14th day of gestation) as well as in the more completely developed choroid plexus (18th day of gestation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of adenylate kinase isoenzymes in rat liver

Total adenylate kinase activity was determined in developing rat liver. The activity was 18 units/g wet weight of tissue in foetal liver; this increased to 41 units/g immediately after birth and continued increasing until adult activities of 150 units/g were reached after two weeks. The adenylate kinase activity was separated into four isoenzymes. Only isoenzymes II and III were observed in foetal rat liver. Isoenzyme II activity was 2 units/g in the foetal liver and increased to 25 units/g in adult liver. Adenylate kinase III activity was 20 units/g in the foetal liver and increased to 118 units/g in adult liver. The possible role that adenylate kinase might have in regulating the energy flow in the developing liver cell is discussed.     

Developmental changes in alcohol-dehydrogenase activity in rat and guinea-pig liver

1. Alcohol-dehydrogenase activity is first detectable in the rat foetus on about the eighteenth day of gestation, after which time it increases to about 25% of the adult activity at birth. Adult activity is reached at about 18 days after birth. The ethanol-oxidizing capacity of liver slices from rats correlates well with the increase of the enzyme activity in vitro. 2. In the guinea pig there is a steady linear increase from about 17 days before term to 5 days after birth. Adult activity is reached between the sixth and eighth postnatal day. 3. Some kinetic properties of liver alcohol dehydrogenase are very similar in newborn and adult rats. 4. Administration of ethanol to pregnant rats during the latter half of gestation had no effect on alcohol-dehydrogenase activity in the liver of the newborn offspring. Intraperitoneal injections of ethanol to newborn and young rats had no effect on the alcohol-dehydrogenase activity of the livers. 5. Intraperitoneal injections of hydrocortisone and triamcinolone to newborn and adult non-adrenalectomized rats had no significant effect on the increase of the alcohol-dehydrogenase activity as studied up to 4 days after the injection.     

Glucocorticoid receptors in developing rat brain and liver

Dexamethasone receptors were measured by conventional equilibrium steroid binding studies in rat liver and brain cytosol, during late prenatal and postnatal development, Receptor binding could be detected in both cytosol preparations as early as the 17th day of gestation. Receptor levels in the cytosol from intact animals reached adult values by the 1st day after birth in both tissues. Using adrenalectomized animals an increase which reached adult values was observed during the first postnatal week for liver and the second postnatal week for brain. At physiological concentrations of endogenous glucocorticoids depletion of receptor from the cytosol of intact animals was minimal at postnatal day 1 and reached adult levels by day 7. chromatographie analysis in DEAE-Sephadex A50 minicolumns of unactivated and activated receptor constituents revealed the same pattern as that of adult animals. Glucocorticoid receptor complex from developing liver and brain was shown to be capable of binding to isolated adult liver nuclei after in vitro activation. However full capacity, for nuclear binding was observed in vivo, after injection of inducing doses of [³H]dexamethasone: By the end of the first week after birth adult nuclear binding capacity was observed in experiments in vivo while values peaked during the second week, in both tissues studied.     

Phenylalanine-pyruvate aminotransferase in immature and adult mammalian tissues induction in fetal rat liver

Normal human fetal liver contains little phenylalanine-pyruvate aminotransferase: between the 11th and 22nd week of gestation its activity (per g) is 8.8% of that in adult liver. In rat liver this enzyme begins to rise a few hours before birth. Precocious increases in the phenylalanine-pyruvate aminotransferase activity of fetal rat liver (but not kidney or brain) were evoked by premature delivery and also by the administration of thyroxine or glucagon in utero. These results, Discussed in relation to related observations on other enzymes, suggest that thyroxine secreted by the fetus, and also another factor relaesed at the beginning of labour, may be the natural stimuli for the developmental formation of phenylalanine-pyruvate aminotransferase.The regulation of hepatic phenylalanine-pyruvate aminotransferase and phenylalanine hydroxylase (L-phenylalanine, tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1) during fetal development is different: in both man and rat, phenylalanine hydroxylase begins to rise earlier and is unaffected by the treatments which enhanced the formation of phenylalanine-pyruvate aminotransferase. In suckling rats (but not in fetuses and adults), an injection of cortisol increased the levels of both enzymes. Hepatocarcinomas of the adult rat were devoid phenylalanine hydroxylase as well as phenylalanine-pyruvate aminotransferase. However, suppression in vivo by substrate analogues (α-methylphenylalanine and p-chlorophenylalanine) was unique for phenylalanine hydroxylase.     

The quantitative determination of phenylalanine hydroxylase in rat tissues. Its developmental formation in liver

A sensitive method was developed for determining the phenylalanine hydroxylase activity of crude tissue preparations in the presence of optimum concentrations of the 6,7-dimethyl-5,6,7,8-tetrahydropterin cofactor (with ascorbate or dithiothreitol to maintain its reduced state) and substrate. Tissue distribution studies showed that, in addition to the liver, the kidney also contains significant phenylalanine hydroxylase activity, one-sixth (in rats) or half (in mice) as much per g as does the liver. The liver and the kidney enzyme have similar kinetic properties; both were located in the soluble phase and were inhibited by the nucleo-mitochondrial fraction. Phenylalanine hydroxylase, like most rat liver enzymes concerned with amino acid catabolism, develops late. On the 20th day of gestation, the liver (and the kidney) is devoid of phenylalanine hydroxylase and at birth contains 20% of the adult activity. During the second postnatal week of development, when the phenylalanine hydroxylase activity was about 40% of the adult value, an injection of cortisol doubled this value. Cortisol had no significant effect on phenylalanine hydroxylase in adult liver or on phenylalanine hydroxylase in kidney at any age.     

Ontogeny of glucocorticoid receptors in rat liver.

Specific receptors for glucocorticoids are present in liver cytosols of rat fetuses at least as early as the 18th day of gestation. The concentration of the receptor begins to decline after the 20th day reaching undetectable levels shortly before and after parturition. The receptor can be detected again 1 to 2 hours after birth, and its levels increase markedly to higher than adult values between the 2nd and 5th day. The reason for the failure to detect specific hormone binding near parturition appears to be due to occupation of binding sites by endogenous steroids rather than the absence of the receptor. This is indicated by the demonstration of both cytoplasmic and nuclear receptor sites in liver slices of newborn rats incubated with labeled dexamethasone at 37 degrees. The cytoplasmic receptors of fetal and adult liver differ in their relative affinity for cortisol and corticosterone. The fetal receptors have a higher affinity for corticosterone than cortisol while the reverse is true for the adult receptors. These observations suggest either the existence of dissimilar receptors in fetal and adult liver or the presence of more than one type of receptor sites. It is therefore possible that subtle differences in the nature of hepatic receptors may be partly responsible for the maturation-dependent qualitative differences in tissue responsiveness to glucocorticoids.     

Cytosolic and mitochondrial isoenzymes of branched-chain amino acid aminotransferase during development of the rat.

The isoenzymic forms of branched-chain amino acid aminotransferase in mitochondria of rat tissues were compared with the better-known cytosolic forms in order to find any regular pattern of expression of these isoenzymes during development. Mitochondria of all tissues examined except brain contained only a type-I isoenzyme differing from the cytosolic type-I isoenzyme in heat stability and activation by mercaptoethanol. Foetal and adult brain mitochondria contained isoenzymes type III as well as type I. The large excess of type-I isoenzyme in foetal liver was localized in mitochondria, apparently of haematopoietic cells. The activity of this isoenzyme declined precipitously (by 80%) from day 19 of gestation at the same period and rate as does the volume fraction of haematopoietic cells that are then leaving the liver. Cortisol treatment accelerated the loss of these cells, and proportionally accelerated loss of the mitochondrial isoenzyme I. A development succession of type-I isoenzyme by the unique type II of liver parenchymal cell cytosols could not be demonstrated, since small, about equal, amounts of types I and II were always present in cytosols of foetal and adult liver. Developmental succession of isoenzymes within tissues was limited to cytosols and was demonstrated by the presence of cytosolic isoenzyme III in foetal and newborn skeletal muscle and kidney, organs which contain only isoenzyme I in the adult.     

Metallothionein synthesis in foetal, neonatal and maternal rat liver

The synthesis of hepatic metallothionein relative to other cytosol proteins was measured by [35S]cysteine incorporation in foetal, neonatal and pregnant rats. The relative rate of hepatic metallothionein synthesis reached a maximum in foetal liver on days 18-21 of gestation. Metallothionein synthesis then declined until weaning, when adult levels were established. The rate of metallothionein synthesis was greater in pregnant rats at term than in nulliparous rats. To determine if circulating inducing agents could play a role in the regulation of metallothionein synthesis in foetal liver we treated pregnant rats with inducers at a time prior to the normal rise in foetal liver metallothionein synthesis. Injections of copper, cadmium or hydrocortisone to 17-day-pregnant dams failed to induce foetal metallothionein synthesis. In contrast, zinc injection to the dam was an effective inducer in the foetuses. Maternal laparotomy (performed to expose the foetus for direct injection of inducers) induced foetal metallothionein synthesis. Metallothionein synthesis in the livers of 17-day-gestation dams was induced by all metal injections and laparotomy but, surprisingly, not by hydrocortisone injection. Maternal adrenalectomy did not influence the subsequent normal elevation in foetal or maternal metallothionein synthesis. These results, in conjunction with previous reports, suggest that mobilization of zinc in serum during late gestation may regulate foetal and maternal changes in metallothionein synthesis.     

Modulation of alkaline phosphodiesterase I in cultured rat hepatocytes

Alkaline phosphodiesterase I activity was measured in adult and foetal rat hepatocytes maintained in primary culture under various conditions. This enzyme was found to be expressed in both cell populations and could be resolved into two bands having apparent molecular weights of 130,000 and 250,000, respectively. Alkaline phosphodiesterase I activity was already at high levels in 15 day foetal liver and, as early as the 19th day of gestation, it reached adult levels. Alkaline phosphodiesterase I levels were well maintained during culture. In the absence of serum, its level continued to increase with time in foetal cells. It dramatically increased by days 4 and 5, in adult cells maintained on fibronectin and plastic, respectively. Dexamethasone stimulated alkaline phosphodiesterase I activity after a lag phase of 8 h, with a maximum reached after 40 h. As this induction was prevented by addition of actinomycin D or cycloheximide, it could be concluded that it required RNA and protein synthesis. Only the major Mr 250,000 form responded to dexamethasone and was sensitive to serum.     

Expression of aldolase isozyme mRNAs in fetal rat liver

The regulation of aldolase isozyme expression during development was studied by measuring the concentrations of mRNAs coding for aldolase A and B subunits in fetal and adult rat liver. Poly(A)-containing RNAs were extracted from livers at various stages of development of fetal rats, and the aldolase A and B subunits in the in vitro translation products of these RNAs were analyzed immunologically. The content of aldolase B mRNA in 14-day fetal liver, measured quantitatively as translational activity, was somewhat smaller than that of aldolase A mRNA; immunologically precipitable aldolase B and A amounted to 0.06% and 0.25% respectively, of the total products. Similar experiments using RNAs from fetuses at later stages, however, showed that aldolase B mRNA increased during development, whereas aldolase A mRNA decreased. In newborn rat liver, aldolase B constituted 0.56% of the total translation products of mRNA, but there was little detectable aldolase A (0.03%). The changes of aldolase mRNA levels were analyzed further by northern blot and dot-blot hybridization experiments using cloned aldolase A and B cDNAs. The content of aldolase B mRNA increased in the fetal stage, and that in newborn rat liver was about 12 times that in 14-day fetal liver. In contrast, the aldolase A mRNA content decreased during gestation and that in newborn rat liver was about one-eighth of that in 14-day fetal liver. These observations suggest that the switch of aldolase isozyme expression in fetal liver is controlled by the levels of the respective mRNAs.     

Changes of gamma-glutamyltranspeptidase activity in the rat during development and comparison of the fetal liver, placental and adult liver enzymes

Changes in the activity of gamma-glutamyltranspeptidase (GGT, EC 2.3.2.2) during development in rats were investigated. The activity of GGT in fetal liver increased rapidly immediately before birth, reached a maximum at birth and then decreased rapidly within a week after birth to nearly the level in adult rat liver. In contrast, placental GGT showed higher activity at an early stage (from day 13 to day 15) of the gestation period, but the activity decreased in the last part of fetal life. The activity in the amniotic fluid increased significantly just before birth, in parallel with the increase of activity in the fetal liver. No change of activity in the uterine wall was observed throughout gestation. The kinetic and immunological properties of partially purified GGTs from fetal liver and placenta were almost identical with those of adult liver GGT. However, the activity of soluble GGT in fetal liver was less effectively inhibited by antibody against adult kidney GGT. Thus, it is likely that at least one isozyme of GGT is present in the soluble fraction of fetal liver.     

Investigation of thyroxine monodeiodinase activity in the liver, kidney and brown adipose tissue of foetal and neonatal rabbit

The maturation of the 5'- and 5-monodeiodinase system in liver, kidney and brown adipose tissue of rabbits, during the foetal period (from 21 days of gestation to birth) and the neonatal period (from birth to 3 weeks of life) was studied. A sudden increase of 5'- and 5-monodeiodinase activity in liver and kidney 3 days before birth was observed, falling to a nadir at day 3 after birth. Foetal and neonatal serum T4, T3 and rT3 concentration were very low and rose progressively with age, reaching adult values at about day 21. In the foetal brown adipose tissue high 5'-monodeiodinase and low 5-monodeiodinase activity was found. The 5'-monodeiodinase decreased during the first days of life whereas the 5-monodeiodinase activity remained at a low stable level until day 3 when the activities of both enzymes increased. The increase of conversion rate of T4 to T3 and rT3 in liver and kidney well correlate with the triiodothyronines concentration in serum from day 3 after birth.     

Regulation of tyrosine aminotransferase in foetal rat liver.

A specific tyrosine aminotransferase, separate from the aspartate aminotransferases, is present in low concentration in foetal rat liver at the 21st day of gestation. Intraperitoneal injections of tyrosine methyl ester into the foetuses in utero increase the activity 2-fold, whereas glucose injections decrease it. Tyrosine, dexamethasone and dibutyryl cyclic AMP induce the enzyme activity in organ culture to the same extent as in adult rat liver in vivo.     

Pteridine cofactor of phenylalanine hydroxylase in fetal rat liver

1. Pteridine cofactor of phenylalanine hydroxylase (EC 1.14.16.1) and dihydropteridine reductase (EC 1.6.99.7) in the phenylalanine hydroxylating system have been studied in the fetal rat liver. 2. Activities of pteridine cofactor and dihydropteridine reductase were measured as about 6 and 50%, respectively, of the levels of adult liver in the liver from fetuses on 20 days of gestation, at this stage the activity of phenylalanine hydroxylase was almost negligible in the liver. 3. Development of the activity of sepiapterin reductase (EC 1.1.1.153), an enzyme involved in the biosynthesis of pteridine cofactor, was studied in rat liver during fetal (20-22 days of gestation), neonatal and adult stages comparing with the activity of dihydrofolate reductase (EC 1.5.1.3). Activities of the enzymes were about 80 and 50%, respectively, of the adult levels at 20 days of gestation. 4. Some characteristics of sepiapterin reductase and dihydropteridine reductase of fetal liver were reported.     

Liver glycogen synthase in the developing foetal rat

Kinetic constants for liver glycogen synthase (UDPglucose: glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11) with respect to UDPglucose have been measured in foetal liver homogenates from samples taken during late gestation (days 17-22) and the first hours after birth. The V of the inactive form of glycogen synthase increased markedly in this period and there was a significant increase in V of the active enzyme to a maximum at day 20 of gestation. The Km for UDPglucose measured in the presence of glucose-6-P (total activity) did not vary greatly, mean values of 0.51 +/- 0.04 mM. Values derived for the inactive enzyme were almost identical. In contrast, Km values for active glycogen synthase in foetal livers during gestation were significantly higher than those for adult liver. Highest values were seen at day 19 of gestation (1.84 +/- 0.08 mM) followed by a steady fall to 0.55 +/- 0.05 mM in the newborn compared with a mean value of 0.48 +/- 0.04 mM for adult liver. Existence of a reduced affinity of active glycogen synthase for UDPglucose must be recognized when assaying the enzyme in foetal liver, particularly when extrapolating values to rates of glycogen synthesis in vivo. Data were obtained only after removal of an amylase-like contaminant from foetal liver samples which invalidated the radioassay of glycogen synthase. This work illustrates the care needed in the analysis of foetal tissue and the interpretation of resulting data when utilizing methods developed for adult tissue.